Extensive clinical and experimental evidence strongly indicates that the complement system plays a critical role in the pathogenesis of a diverse group of human diseases that includes some of the hemolytic anemias and male infertility. Given the complex and chronic nature of these diseases in which a pathogenic role of complement still needs to be defined, molecular engineered mouse models that we propose to use in this application provide powerful tools to conduct experiments that would be impossible to perform in humans. Through three different activation pathways and formation of the membrane attack complex (MAC), the complement system mediates both acquired and innate responses to defend against bacterial infection, and to dispose of immune complexes or apoptotic cells. Activated complement and MAC can also damage self cells and thereby mediate pathological responses leading to chronic diseases. An array of complement regulatory proteins including CD59, a key inhibitor of MAC formation, have evolved to protect self cells from complement damage, which can result from either increased complement activation with MAC formation or decreased regulation. Development of adequate animal models to study the relative role of the MAC and its regulation in the pathogenesis of human diseases has been hampered because of the unexpected presence of two Cd59 genes (termed mCd59a and mCd59b) in the mouse genome. Preliminary studies on mCd59a and mCd59b knockout mice demonstrated that the mCd59ab double knockout mouse (mCdSgab-/-), which we have successfully generated by targeted deletion of both genes, was required to investigate the role of MAC regulation in the pathogenesis of human diseases. Our hypothesis is that absence of CD59 function will result in MAC-mediated tissue damage and focal pathology in different organs. Based on the phenotypic features observed in mCdSgb-/-' and mCdSgab-/- mice, we will study mCdSgab'-/- mice focusing on two phenotypic traits representative of human diseases in which a role of complement has long been suspected: 1) complement-mediated hemolytic anemia with platelet activation; and 2) male infertility. Using available C3 or C4 deficient mice, we will differentiate specific roles of complement and its activation pathways from complement-independent functions of CD59. Using transgenic mice that express hCD59 only in RBC and crossing them with mCdSgab-/-, we will verify the causal relationships between the hemolytic anemia and the loss of CD59 in mCd59ab-/-. Successful accomplishment of our goals will define the role of MAC and its regulation in three paradigmatic human diseases, and will provide the research community with a much-needed model to continue studying the role of complement in human pathology.